Pcl5-GFP is transported into the S. cerevisiae nucleus. (A) Nuclear localization of high and low amounts of the functional cyclin Pcl5-GFP. Localization was analyzed in S. cerevisiae cells (RH3239) expressing Pcl5-GFP fusion protein derived from its endogenous PCL5 promoter at the chromosomal locus or pcl5 mutant cells (RH3238) expressing Pcl5-GFP fusion protein from the MET25 promoter on a low-copy (CEN) plasmid (pME2844) or on a high-copy (2μm) plasmid (pME2846). Cells were grown to early log phase at 30°C and analyzed by DIC microscopy, DAPI staining, and fluorescence microscopy (GFP). “X/200” in parentheses means “X” number of 200 cells expressing PCL5-GFP detectable in the nucleus. (B) Nuclear localization of Pcl5-GFP is independent of the availability of amino acids or the proteins Gcn4, Pho85, or Pho81. Nuclear localization of Pcl5-GFP expressed from the MET25 promoter on a high-copy plasmid (pME2846) in pcl5 (RH3238) under amino acid starvation conditions (−Leu), pho85 (RH3242), gcn4 (RH1408), and pho81 (RH3241) mutant strains. Yeast cells were grown and analyzed as described above. Leucine starvation (−Leu) was induced in leucine-auxotrophic yeast cells by shifting to synthetic medium lacking leucine.

Nuclear localization of yeast Pcl5 is required for Gcn4 degradation. (A) PCL5-GFP-NES is correctly expressed as 54-kDa equivalent to PCL5-GFP. Yeast pcl5 cells (RH3238) were transformed to express either PCL5-GFP (pME2846) or PCL5-GFP-NES (pME2861) from the high-copy plasmids under the control of the MET25 promoter. Cells were grown to early log phase, and the expression of the GFP fusion proteins was analyzed by Western blotting with monoclonal anti-GFP antibodies. (B) Pcl5-GFP-NES is transported out of the nucleus into the cytoplasm. Localization of the fusion proteins Pcl5-GFP (pME2846) and Pcl5-GFP-NES (pME2861) expressed under the control of the MET25 promoter on high-copy (2μm) plasmid was analyzed in a pcl5 mutant strain (RH3238) by fluorescence microscopy (GFP), DAPI staining, and DIC microscopy. The numbers in parentheses represent the total amount of cells investigated (n = 200) and the respective amount showing the displayed localization. (C) Pcl5-GFP-NES is incapable of suppressing the toxicity of overexpressed GCN4 in the absence of a functional PCL5 gene. Wild-type cells (RH3237) and pcl5 mutant cells (RH3238) expressing GAL1-driven myc³-GCN4 from the low-copy plasmid pME2848 alone or together with GFP (pME2849), PCL5-GFP (pME2846), or PCL5-GFP-NES (pME2861) under the control of the MET25 promoter on a high-copy plasmid were spotted in 10-fold dilutions on glucose (repressing conditions) and galactose (inducing conditions) to induce expression of GCN4 driven by the GAL1 promoter. As controls, wild-type and pcl5 mutant cells transformed with the empty vector were used (control). The given doubling times are the average of at least three independent growth tests in liquid culture with glucose (repressing conditions) or galactose (inducing condition) as a carbon source. (D) Pcl5-GFP-NES is unable to induce Gcn4 degradation. The same wild-type and pcl5 yeast strains as in panel C were transformed to express myc³-GCN4 from plasmid pME2848 alone or together with PCL5-GFP (pME2846) or PCL5-GFP-NES (pME2861). Protein levels of myc³-Gcn4 or Cdc28 were determined at the indicated time points after the shift to glucose medium. The numbers given below each lane indicate the remaining Gcn4-percentage compared to Cdc28 as an internal standard.

The C-terminal part of Pcl5 directs nuclear localization. (A) Yeast pcl5 mutant strain RH3238 was transformed to express either GFP alone (pME2849) or in N-terminal fusion with Pcl5_aa1-229 (pME2846), Pcl5_aa1-95 (pME2850), Pcl5_aa1-127 (pME2851), Pcl5_aa1-180 (pME2853), Pcl5_aa61-229 (pME2854), Pcl5_aa111-180 (pME2855), Pcl5_aa111-229 (pME2856), Pcl5_aa153-229 (pME2857), Pcl5_aa181-229 (pME2859), Pcl5_aa204-218 (pME2950), Aro7-Pcl5_aa207-215 (pME2951), Pcl5_aa181-229*** (with the mutated NLS motif marked by asterisks) (pME3578), Pcl5_aa1-229*** (pME3577), Pcl5_aa61-229*** (pME3370), Pcl5_aa61-180 (pME2858), Pcl5_aa79-178 (pME3574), Pho80_aa1-73-Pcl5_aa79-178-Pho80_aa170-294 (pME2860), or Pho80_aa1-73-Pcl5_aa79-178 (pME2948) from the MET25 promoter on a high-copy plasmid. In addition, Pho80_aa1-73-Pcl5_aa79-178 was expressed from a low-copy plasmid under the control of the PCL5 promoter (pME3576). GFP signals were analyzed by fluorescence microscopy (GFP) or DIC microscopy. Nuclear localization is indicated by white colored arrows. An arrowhead only marks plasma membrane localization. Accumulation in the cytoplasm is highlighted by a rendered blank arrow. A dashed arrow is used in the case of cytoplasmic aggregates. The differences in sizes of the cells shown are due to different enlargement. “X/200” in parentheses represents the fraction “X” of 200 cells displaying the marked staining pattern. (B) Yeast pcl5 cells transformed with the same high-copy plasmids as in panel A beside pME2849 encoding GFP alone and the low-copy plasmid pME3576 expressing Pho80_aa1-73-Pcl5_aa79-178 were grown to early log phase, and the expression of the GFP fusion proteins was analyzed by immunoblotting with monoclonal anti-GFP antibodies. Protein extracts of the cells are blotted in the same order as in panel A (left panel, top to bottom; right panel, top to bottom).

The mutation of the β-importin encoding KAP95 prevents the import of Pcl5 into the nucleus. (A) Nuclear import of a functional Pcl5-GFP fusion protein expressed from the high-copy plasmid pME2846 under the control of the MET25 promoter was analyzed in five temperature-sensitive importin mutant strains by fluorescence microscopy (GFP) and DIC microscopy. Pcl5 translocation is not affected in the mutant strains mtr10 (RH2701), kap104 (RH2702), pse1 (RH2703), or pse1/kap123 (RH2706), whereas the kap95 (RH2704) mutation impairs the uptake of Pcl5-GFP at the restrictive temperature of 30°C. Expression of KAP95 under the control of the MET25 promoter on a high-copy plasmid (pME3583) in kap95 mutant cells (RH2704) restores the uptake of Pcl5-GFP to the nucleus (∼ 40%) at 30 and 37°C. Furthermore, five mutant strains with the nonconditional importin mutations kap114 (RH3058), kap123 (RH2707), nmd5 (RH2708), pdr6 (RH2709), and sxm1 (RH2710) were examined for subcellular localization of Pcl5-GFP by fluorescence microscopy. Nuclear import of Pcl5 was unaffected in all five mutant strains and was indistinguishable from that in the wild-type (wt) control (RH3237). (B) Protein-protein interaction of Pcl5 with Kap95. Yeast pcl5 mutant strain RH3238 was transformed to express either myc⁹-PCL5 (pME2865) with GST (GST on pYGEX-2T), GST-KAP104 (pME3447), or GST-PSE1 (pME3448) as negative controls or myc⁹-PCL5 (pME2865), together with GST-PHO85 (pME2866), as a positive control. In addition, yeast strain RH3238 was transformed to express myc⁹-PCL5 (pME2865), together with GST-KAP95 (pME3372). Protein levels of the fusion proteins were determined by Western blotting with rabbit anti-GST and mouse anti-myc antibodies before and after incubation with glutathione-Sepharose. The left part (Input) represents the GST, GST-Kap95, GST-Kap104, GST-Pse1, and myc⁹-Pcl5 before glutathione-Sepharose incubation to ensure that the initial protein extracts contain similar amounts of the fusion proteins. On the right (Beads) the elutions of the glutathione beads are shown. The GST fusion proteins are marked with an arrow in the respective lane of the elutions. (C) Mutation of the C-terminal NLS motif in Pcl5 does not impede the interaction to Kap95. This is the same experiment as shown in panel B. Yeast pcl5 mutant strain RH3238 was transformed to express either myc⁹-PCL5 (pME2865) together with GST (GST on pYGEX-2T), GST-PHO85 (pME2866), or GST-KAP95 (pME3372). In addition, pcl5 mutant cells (RH3238) were transformed to express myc⁹-PCL5*** with a mutated NLS motif (pME3577), together with GST (GST on pYGEX-2T) or GST-KAP95 (pME3372). Input (left part) represents the initial protein extracts before glutathione-Sepharose incubation. On the right side (Beads) the elutions of the beads are shown.

Functional analysis of Pcl5-Pho80 hybrids fused to GFP. (A) The Pho80-Pcl5_aa79-178-Pho80 hybrid is able to suppress the overexpression toxicity of Gcn4 like full-length-Pcl5 in pcl5 yeast cells. Wild-type cells (RH3237) and pcl5 mutant cells (RH3238) expressing myc³-GCN4 from the GAL1 promoter (pME2848), together with MET25-PCL5_aa61-229-GFP (pME2854, 2μm), MET25-PCL5_aa61-180-GFP (pME2858, 2μm), MET25-PCL5_aa1-180-GFP (pME2853, 2μm), MET25-PCL5_aa1-180-GFP (pME3573, CEN), MET25-PHO80_aa1-73-PCL5_aa79-178-PHO80_aa170-294-GFP (pME2860, 2μm), or MET25-PHO80_aa1-73-PCL5_aa79-178-GFP (pME2948, 2μm), and PCL5prom-PHO80_aa1-73-PCL5_aa79-178-GFP (pME3576, CEN) were spotted in 10-fold dilutions on glucose and galactose to induce expression of GCN4 driven by the GAL1 promoter. The plates were incubated for 3 days at 30°C. Furthermore, these cells were used for growth tests in liquid culture with glucose or galactose as a carbon source, and the doubling times of three or more independent cultures were determined. (B) The Pho80-Pcl5_aa79-178-Pho80 hybrid is able to promote Gcn4 degradation like full-length Pcl5 in pcl5 yeast cells. Protein levels of myc³-Gcn4, Cdc28, or eIF-2 were determined by Western blotting at the indicated time points after the GAL1 promoter shutoff in the same transformed yeast cells as described in panel A. The numbers given below each lane indicate the remaining Gcn4 percentage compared to Cdc28 or eIF-2 as internal standard quantified with a Kodak imaging station of the gel shown.

Domain analysis of yeast cyclin Pcl5. (A) On the left side of the panel are listed Pcl5 fragments fused to the N terminus of GFP. pME2846 (Pcl5_aa1-229), pME3577 (Pcl5_aa1-229*** containing the three amino acid substitutions K209A, R210A, and R212A marked by asterisks), pME2850 (Pcl5_aa1-95), pME2851 (Pcl5_aa1-127), pME2853 (Pcl5_aa1-180), pME2858 (Pcl5_aa61-180), pME2854 (Pcl5_aa61-229), pME3370 (Pcl5_aa61-229*** containing the three amino acid substitutions K209A, R210A, and R212A), pME2855 (Pcl5_aa111-180), pME2856 (Pcl5_aa111-229), pME2857 (Pcl5_aa153-229), pME2859 (Pcl5_aa181-229), pME3578 (Pcl5_aa181-229*** containing the three amino acid substitutions K209A, R210A, and R212A), pME3574 (Pcl5_aa79-178), pME2860 (Pho80_aa1-73-Pcl5_aa79-178-Pho80_aa170-294), pME2948 (Pho80_aa1-73-Pcl5_aa79-178), and pME2849 (GFP alone). A summary of the results is shown on the right. The columns indicate the subcellular localization of the different PCL5 or PCL5-PHO80 deletions (Fig. 3) and their ability to complement the pcl5 phenotype of Gcn4 toxicity or to promote Gcn4 degradation (Fig. 5). CB, cyclin box; N, nuclear; PM, plasma membrane; C, cytoplasm; +, yeast cells are able to complement the pcl5 phenotype or to degrade Gcn4, respectively; −, yeast cells are not able to complement the pcl5 phenotype or rather to degrade Gcn4. (B) Scheme of identified Pcl5 domains. The relative positions of the different domains within the full-length Pcl5 protein are shown. The region from aa 1 to 95 represents a putative plasma membrane binding site motif, and the middle part consisting of aa 79 to 178 is required for the right substrate specificity. The Pcl5 carboxyl terminus of aa 207 to 215 is required for nuclear localization. CB, cyclin box.